Ysabel Santos

Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray

ABSTRACT We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with <20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

Homology of Vibrio anguillarum strains causing epizootics in turbot, salmon and trout reared on the Atlantic coast of Spain

Abstract In this paper we report the physiological, biochemical, serological, and molecular properties of pathogenic Vibrio anguillarum strains isolated from turbot, salmon and trout reared at several seawater hatcheries on the Galician coast (N.W. Spain). These characteristics were compared with reference strains isolated from different fish or in other geographic areas. The role of extracellular virulence factors in the pathogenesis of vibriosis was also investigated. Agglutination reactions and LPS patterns revealed that most of our isolates belonged to serotype 1, and only the strain responsible for a turbot epizootic in 1985 belonged to serotype 2. The main differential phenotypic traits between these pathogenic vibrios were indol reaction, growth at 37°C and acid production from arabinose, galactose and sorbitol. All the isolates displayed strong protease, amylase, and phospholipase activities, and produced haemolysins against human, sheep and trout erythrocytes. In addition, these strains exhibited similar drug resistance patterns, being sensitive to nitrofuranes, flumequine, oxolinic acid, oxytetracycline and chloramphenicol, and highly resistant to ampicillin. Although the extracellular products from our Vibrio isolates displayed strong cytotoxic activity on the five fish cell lines tested, a non-pathogenic reference strain also showed a positive toxic effect, which indicates that a relationship between fish virulence and cytotoxicity cannot be established for all V. anguillarum strains. Whereas the Northwest Spain isolates belonging to serotype 1 shared one plasmid (47 Md) similar to the virulence plasmid pJM1, in the pathogenic vibrios assigned to serotype 2, no plasmids were detected and, hence, their virulence properties are chromosome-coded.

Tenacibaculum discolor sp. nov. and Tenacibaculum gallaicum sp. nov., isolated from sole (Solea senegalensis) and turbot (Psetta maxima) culture systems

Two Gram-negative, rod-shaped, gliding bacterial strains, designated strain LL04 11.1.1(T) and strain A37.1(T), were isolated from a diseased sole (Solea senegalensis) and from seawater from a holding tank for turbot (Psetta maxima), respectively. The strains grew on solid media as bright yellow colonies with uneven edges; the colonies did not adhere to the agar. The bacteria were able to grow at temperatures in the range 14 to 38 degrees C and from pH 6.0 to pH 8.0. The DNA G+C contents of strains LL04 11.1.1(T) and A37.1(T) were 32.1 and 32.7 mol%, respectively. Analysis of the 16S rRNA gene sequences indicated that strains LL04 11.1.1(T) and A37.1(T) were members of the genus Tenacibaculum in the family Flavobacteriaceae. The sequence similarities of the two isolates with respect to the type strains of recognized members of the genus ranged from 94.2 to 99.4%. DNA-DNA hybridization studies revealed that the strains studied represent two distinct novel species of the genus Tenacibaculum, for which the names Tenacibaculum discolor sp. nov. [type strain LL04 11.1.1(T) (=NCIMB 14278(T)=DSM 18842(T))] and Tenacibaculum gallaicum sp. nov. [type strain A37.1(T) (=NCIMB 14147(T)=DSM 18841(T))] are proposed.

Tenacibaculum soleae sp. nov., isolated from diseased sole (Solea senegalensis Kaup)

A novel Gram-negative, rod-shaped, gliding bacterial strain designated LL04 12.1.7T was isolated from diseased sole (Solea senegalensis Kaup) in Galicia, Spain. Colonies were yellow-pigmented with uneven edges and did not adhere to the agar. The DNA G+C content of the strain was 29.8 mol%. 16S rRNA gene sequence similarity analysis indicated that strain LL04 12.1.7T is a member of the genus Tenacibaculum in the family Flavobacteriaceae. Sequence similarities between the isolate and the type strains of other members of the genus were 96.7-94.8 %. The major fatty acids (>10 % of total fatty acids) were iso-C15 : 0 (23.1 %), iso-C15 : 0 3-OH (10.6 %), C15 : 1 omega 6c (12.2 %) and summed feature 3 (comprising C16 : 1 omega 7c and/or iso-C15 : 0 2-OH, 11.0 %). Genotypic and phenotypic data distinguished strain LL04 12.1.7T from the 11 recognized Tenacibaculum species, indicating that it represents a novel species, for which the name Tenacibaculum soleae sp. nov. is proposed. The type strain is strain LL04 12.1.7T (=CECT 7292T =NCIMB 14368T).

Usefulness of the API-20E system for the identification of bacterial fish pathogens.

Abstract A total of 223 isolates belonging to motile Aeromonas, A. salmonicida, Vibrio anguillarum, Pasteurella piscicida and Yersinia ruckeri species were tested by the API-20E system and the results compared with those obtained with standard biochemical tube and plate tests. Depending on the species, the API-20E yielded false negative and positive reactions for fermentation of different sugars, lysine decarboxylase (LDC), Voges Proskauer (VP), citrate and gelatinase reactions. Thirteen of 32 (41%) A. salmonicida strains and 9 of 53 (17%) Y. ruckeri isolates were correctly identified using the API index. Forty-five of 69 (65%) motile Aeromonas tested (including 34 A. hydrophila, 10 A. sobria and 1 A. caviae strains) were identified as A. hydrophila. The low discrimination profiles generated by 9 Yersinia and 2 motile Aeromonas isolates were avoided using the supplementary tests cited in the API index. In the case of V. anguillarum and P. piscicida, which are not at present included in the API-20E system, 35 of 53 strains of V. anguillarum were wrongly identified as A. hydrophila, and all the P. piscicida isolates were incorrectly identified as Pseudomonas fluorescens/Ps. putida. A large number of isolates, including some reference strains, produced uncoded profiles. From these results, we consider that the API-20E system will be a useful tool for field diagnosis of bacterial fish diseases if its database is expanded to include V. anguillarum, P. piscicida, A. sobria and A. caviae species and the new profiles generated by strains of species already considered. In addition, some reactions necessary to discriminate between strains which share the same profile number are proposed.

Vaccination trials on gilthead seabream (Sparus aurata) against Pasteurella piscicida

A comparative study of the efficacy of two vaccine formulations, a whole-cell bacterin (WCB) and a toxoid-enriched whole-cell vaccine (WCEB), against Pasteurella piscicida was conducted by bath immersion in gilthead seabream in order to evaluate the role of the extracellular products (ECP) as protective antigens against this pathogen. With this aim, two strains showing differences in their ECP composition were used to prepare both vaccines. Only the toxoid-enriched vaccine conferred protection against P. piscicida within a 4-week period. The relative percent survival (RPS) acheived with this type of vaccine ranged between 37 and 41 depending on the bacterial strain and dose used in the challenge. Although this protection level is not very high, we consider that it is valuable considering the economic importance of the fish susceptible to pasteurellosis throughout the world. The booster immunization with the WCEB P. piscicida formulation did not increase the protection levels of gilthead seabream. The antibody response in the sera of both immunized fish groups was very low with no correlation between the level of agglutinating antibodies and the protection. In addition, this vaccine did not confer cross-protection against serotypes 01 and 02 of Vibrio anguillarum.

Looking for New Targets: Simple Coumarins as Antibacterial Agents

The dramatic worldwide increase of dangerous infections by resistant and multi-resistant microbes makes the search of new molecules and new chemical entities an important topic in Medicinal Chemistry. As the ideal drug candidate has not been attained, an intensive search for new and innovative antimicrobials is still needed. A small series of 3-amino/nitrocoumarins without substitutions or substituted by methyl or methoxy groups at different positions were synthesized and evaluated for their antibacterial and antifungal activities against clinical isolates of Staphylococcus aureus, Escherichia coli and Candida albicans strains. Some of these structurally simple molecules exhibited antibacterial activity. The preliminary SAR study showed that the antibacterial activity against E. coli and S. aureus was dependent on the kind and position of the substitution pattern at the coumarin moiety.

An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish

A hitherto unrecognised lethal toxin from the extracellular products (ECP) of Aeromonas hydrophila is described. The pure toxin was 300 times more toxic than the crude ECP and is the most toxic substance so far described from this bacterium, with a minimum lethal dose of 0.05 micrograms g-1 fish. The toxin had high acetylcholinesterase activity and occurred in native ECP as a monomeric 15.5 kDa polypeptide. The purified toxin had five isoelectric focusing forms ranging from pl 4.45 to 4.70. The ECP of each of six strains of A. hydrophila isolated from fish possessed acetylcholinesterase activity suggesting that the toxin is common in this species. The toxin was not a cytolysin and produced no gross pathology in injected fish. Its enzymic nature, low lethal dose, lack of tissue pathology and its apparent narcotic effect suggest that this toxin may act upon the central nervous system of the fish.

Synthesis and structure-activity relationships of novel amino/nitro substituted 3-arylcoumarins as antibacterial agents.

A new series of amino/nitro-substituted 3-arylcoumarins were synthesized and their antibacterial activity against clinical isolates of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) was evaluated. Some of these molecules exhibited antibacterial activity against S. aureus comparable to the standards used (oxolinic acid and ampicillin). The preliminary structure-activity relationship (SAR) study showed that the antibacterial activity against S. aureus depends on the position of the 3-arylcoumarin substitution pattern. With the aim of finding the structural features for the antibacterial activity and selectivity, in the present manuscript different positions of nitro, methyl, methoxy, amino and bromo substituents on the 3-arylcoumarin scaffold were reported.